Figure 2. TGF-β3 was expressed during lip formation and up-regulated during fusion of surgically sutured cleft lip. The upper lip of the unaffected CL/Fr mouse embryo begins to fuse at E12. Immunohistochemistry showed juxtaposing surface epithelia of the lips immunopositive for TGF-β3 (a) as detected by the russet color positive reaction. TGF-β3 was not detected in the surface epithelium of the cleft lip (b). In the E16 fetus with cleft lip that was surgically repaired, a large number of platelets (black arrowheads) was observed populating the site of operation 30 min after surgery (c). These platelets were immunopositive for TGF-β3. Some endothelial cells were also positive for TGF-β3 (inset in c). In control unoperated clefts, tissues remained immunonegative for TGF-β3 (d). Sections were counterstained with hematoxylin. Asterisks indicate oral epithelium. Scale bars in (a-d) are 100 µm. The average number of TGF-β3-positive platelets was 12 + 1.86 (N = 5), whereas none was observed in the control group (N = 5) (e), *p < 0.01. Samples were also collected 10 min after surgery and assayed for TGF-β3 expression level (f). In the sutured group (1.86 + 0.21, N = 5), the expression of TGF-β3, normalized for β-actin expression level, was 3.4-fold higher than that of the control group (0.55 + 0.13, N = 5), *p < 0.01.